No abstract

Ethylene production and floral senescence following compatible and incompatible pollinations were studied in a self-incompatible species, Petunia inflata. Both compatible and incompatible pollinations resulted in a burst of ethylene synthesis that peaked 3 hours after pollination. P. inflata pollen was found to carry large amounts of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The amount of pollen-held ACC varied in different genetic backgrounds, and the magnitude of the peak correlated with the amount of ACC borne by the pollen. Aminooxyacetic acid (AOA), an inhibitor of ACC synthesis, had no inhibitory effect on this ethylene response, indicating that pollen-borne ACC was largely responsible for the early synthesis of ethylene. After compatible pollination, a second increase in ethylene synthesis began at 18 hours, and the first sign of senescence appeared at 36 hours. Upon treatment with AOA, the second phase of ethylene production was reduced by 95%, indicating that endogenous ACC synthesis was required for this phase of ethylene synthesis. AOA treatment also delayed senescence to 6 days after anthesis. After incompatible pollination, a second increase in ethylene production did not occur until 3 days, and the first sign of senescence occurred 12 hours later. Unpollinated flowers showed an increase in ethylene production 3 to 4 days after anthesis and displayed signs of senescence 1 day later. The significance of the early and late phases of pollination-induced ethylene synthesis is discussed.

Three S-allele-associated proteins (S-proteins) of Petunia inflata, a species with gametophytic self-incompatibility, were previously found to share sequence similarity with two fungal ribonucleases, RNase T2 and RNase Rh. In this study, the S-proteins from P. inflata plants of SS2 and S2S3 genotypes were purified to homogeneity by gel filtration and cation-exchange chromatography, and their enzymatic properties were characterized. The three S-proteins (Si, S2, and S3), with pairwise sequence identity ranging from 73.1 to 80.5%, were similar in most of the enzymatic properties characterized. The ribonuclease activity had a pH optimum of 7.0 and a temperature optimum of 500C. Diethylpyrocarbonate at 1 millimolar almost completely abolished the ribonuclease activity; cupric sulfate and zinc sulfate at 1 millimolar reduced the ribonuclease activity of the three S-proteins by 50 to 75%. EDTA and RNasin had no inhibitory effect. All three Sproteins hydrolyzed polycytidylic acid preferentially, but varied in their nucleolytic activity toward polyadenylic acid and polyuridylic acid.Gametophytic self-incompatibility acts to prevent self-fertilization and consequent inbreeding in many species of the Solanaceae, including Petunia inflata. In Petunia, self-incompatibility is controlled by a single polymorphic locus, the Slocus. The S-allele products of pollen and pistil are presumed to interact in a manner that allows pistils to discriminate between self-and cross-pollen. The result of this specific selfrecognition event is that pollen bearing an S-allele identical to either one of the two alleles carried by the pistil suffers growth arrest, while pollen bearing an allele different from both alleles carried by the pistil is able to grow down the pistil to effect fertilization.S-allele-associated pistil proteins (S-proteins) have been identified in many species with gametophytic self-incompatibility (1, 7, 8, 17, 19-21, 23, 24, 29). The sequences of a number of solanaceous S-proteins have been deduced from the corresponding cDNA sequences (1-3, 10, 20, 30). All the full-length cDNA sequences predict a leader peptide that is not found in the mature protein.

Petunia inflata, a species with gametophytic self-incompatibility, has previously been found to contain a large number of ribonucleases in the pistil. The best characterized of the pistil ribonucleases are the products of the S alleles, the S proteins, which are thought to be involved in self-incompatibility interactions. Here we report the characterization of a gene encoding another pistil ribonuclease of P. inflata, RNase X2. Degenerate oligonucleotides, synthesized based on the amino-terminal sequence of RNase X2, were used as probes to isolate cDNA clones, one of which was in turn used as a probe to isolate genomic clones containing the gene for RNase X2, rnx2. The deduced amino acid sequence of RNase X2 shows 42% to 71% identity to the 20 solanaceous S proteins reported so far, with the highest degree of similarity being to S3 and S6 proteins of Nicotiana alata. The cDNA sequence predicts a leader peptide of 22 amino acids, suggesting that RNase X2, like S proteins, is an extracellular ribonuclease. Also, similar to the S gene, rnx2 is expressed only in the pistil, and contains a single intron comparable in size and identical in location to that of the S gene. However, rnx2 is not linked to the S locus, and, in contrast to the highly polymorphic S gene, it is monomorphic. The possible biological function of RNase X2 is discussed.

We isolated a cDNA clone from a pistil cDNA library of Petunia inflata which encodes a protein, PPT, with sequence similarity to gamma-thionins. Characterization of a genomic clone containing a PPT gene revealed the presence of a single intron. Northern analysis revealed that the PPT gene was predominantly expressed in the pistil during all stages of flower development. Since thionins have been implicated in plant defense against pathogens, PPT may play a role similar to that of other defense-related proteins found in the pistil, defending the pistil against pathogen infection.